- Email: [email protected]
Preventive effects of exogenous phospholipases on inhibition by ferrous ions of [3H]MK-801 binding in rat brain synaptic membranes
Neurochemistry International 34 (1999) 193±201
Preventive eects of exogenous phospholipases on inhibition by ferrous ions of [3H]MK-801 binding in rat brain synaptic membranes Kiyokazu Ogita, Makoto Shuto, Takayuki Manabe, Nobuyuki Kuramoto, Yukio Yoneda* Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-0101, Japan
Abstract Prior treatment with ferrous chloride led to marked inhibition of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 mM in rat brain synaptic membranes. Both phospholipases A2 and C signi®cantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor a-tocopherol aected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated free fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were ®rst treated with fatty acids, followed by the second treatment with ferrous chloride. These results suggest that membrane phospholipids may be at least in part responsible for interference by ferrous ions with opening processes of the native NMDA channel through molecular mechanisms associated with the liberation of unsaturated free fatty acids in rat brain. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Excitatory amino acid receptors are thought to play a key role in mechanisms underlying important physiological functions of the brain such as learning and memory (Collingridge and Singer, 1990; Cotman et al., 1988), in addition to crisis of a variety of acute neurological and chronic neurodegenerative disorders. The latter includes hypoxic-ishemic brain injury, trauma, epilepsy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and AIDS dementia (Choi, 1988; Lipton and Rosenberg, 1994). Early pharmacological studies have revealed that an ionotropic subclass sensitive to N-methyl-D-aspartic acid (NMDA) functions as a receptor ionophore complex consisting * Corresponding author. Tel.: +81-720-66-3109; fax: +81-720-663109. E-mail address: [email protected] (Y. Yoneda)
of at least four distinct domains: (a) an ion channel domain highly permeable to Ca2+ , (b) an NMDA recognition domain with high anity for the endogenous agonist L-glutamic acid (Glu), (c) a glycine (Gly) recognition domain with anity for co-agonists including Gly and D-serine and (d) a polyamine recognition domain with several inde®nite properties. Moreover, opening of the NMDA channel is interfered by endogenous cations including Mg2+ , Zn2+ and H+, in voltage-dependent and independent manners. The NMDA channel is radiolabeled by [3H](+)-5-methyl10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) which only gains access to the binding site located within the channel when it is opened (Lodge and Collingridge, 1991). Moreover, increased intracellular Ca2+ is supposed to be responsible for feedback inhibition of opening processes of the NMDA channel through calmodulin linked to an intracellular C-terminal region within the principal subunit
0197-0186/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 8 ) 0 0 0 8 7 - 4
194
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
NMDAR1 (Ehlers et al., 1996). By contrast, dierent calmodulin antagonists all inhibit (Ogita et al., 1991), but Ca2+ ions stimulate (Enomoto et al., 1992; Han et al., 1995) binding of [3H]MK-801 to the open NMDA channel in rat brain synaptic membranes. In addition to regulation by these endogenous cations, the in vitro addition of Fe2+ but not Fe3+ inhibits [3H]MK-801 binding through mechanisms entirely dierent from those underlying the inhibition by other Fe2+ -containing compounds including sodium nitroprusside and potassium ferrocyanide (Shuto et al., 1997). Iron is an important constituent in the brain at a substantially high concentration as compared with any other metals (Yehuda and Youdim, 1988). Among dierent brain regions, for instance, iron is enriched at a relatively high concentration in the basal ganglia, red nucleus and dentate nucleus (Bradbury, 1997; Hill and Switzer, 1984; Riederer et al., 1989). Iron is shown to play a critical role in the pathogenesis of particular neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, in association with oxidative stress through the generation of oxygen free radicals (Gerlach et al., 1994), which are supposed to be responsible for mediating neuronal injuries associated with iron by way of lipid peroxidation of cell membranes (Floyd and Carney, 1991; Hall and Braughler, 1993; Halliwell, 1992). Indeed, Fe2+ markedly facilitates formation of lipid hydroperoxides with a concomitant loss of cellular viability in cultured spinal neurons (Zhang et al., 1996). Therefore, lipid peroxidation by iron could ignite cascade processes for crisis of a variety of neurological disorders associated with aging (Youdim et al., 1993) which is shown to induce increased accumulation of iron in particular regions of rat brain (Benkovic and Connor, 1993). However, an opposite proposal that ascorbate/Fe2+ -induced oxidative stress leads to increased activities of the NMDA receptor, such as [3H]g-aminobutyric acid release, Na+ in¯ux and [3H]MK-801 binding in cultured retinal cells is available in the literature (Agostinho et al., 1996). Accordingly, the present study was undertaken to investigate possible mechanisms for the inhibition by ferrous chloride of [3H]MK-801 binding to a native ion channel associated with the NMDA receptor in rat brain synaptic membranes. 2. Materials and methods 2.1. Materials [3H]MK-801 ([3-3H]MK-801, 740 GBq/mmol) was purchased from NEN/DuPont (Boston, MA, USA). Arachidonic, behenic, docosahexaenoic, linoleic, oleic and palmitoeic acids, phospholipase A2 (PLA2), phospholipase C (PLC), spermidine (SPD), superoxide dis-
mutase (SOD) and a-tocopherol were all provided by Sigma Chemical (St. Louis, MA, USA). Stearic acid was purchased from Doosan Serdary Research Laboratories (NJ, USA). Arachidic acid, ferrous chloride and palmitic acid were supplied by Wako Chemical (Osaka, Japan). Other chemicals used were all of the highest purity commercially available. All solutions were freshly prepared immediately before each use. 2.2. Membrane preparation Crude synaptic membrane fractions obtained from whole brains (including cerebellum) of adult male Wistar rats weighing 200±250 g were washed once by suspension in 40 volumes of 50 mM Tris-acetate buer (pH 7.4) using a Physcotron homogenizer at a setting no. 6 for 1 min at 48C, followed by centrifugation at 50,000 g for 30 min, as described previously (Enomoto et al., 1992). Resultant pellets were suspended in 8 volumes of the same buer and stored at ÿ808C until use. On the day of the experiment, these frozen suspensions were thawed at room temperature and directly used for binding assays described below as `non-washed' membrane preparations. Buers and any other solutions used in the present study were all ®ltered each time before use through a nitrocellulose membrane ®lter with a pore size of 450 nm to avoid possible microbial contamination (Yoneda and Ogita, 1989). 2.3. Pretreatment `Non-washed' membranes were incubated with either phospholipases or free fatty acids at 308C for 30 min in 50 mM Tris-acetate buer (pH 7.4) at an approximate protein concentration of 0.2 mg protein/ml, followed by centrifugation at 50,000 g for 30 min. The resultant pellets were again suspended in the same volume of buer followed by centrifugation. Pellets thus obtained were suspended in the same volume of buer, and the suspensions were again incubated with ferrous chloride at dierent concentrations for 30 min at 308C, followed by two cycles of washing procedures as described above. In addition, `non-washed' membranes were pretreated with ferrous chloride at dierent concentrations in either the presence or absence of 10 mM a-tocopherol and SOD at 200 and 400 units/ml, followed by two cycles of washing procedures, as needed. The ®nal pellets thus obtained were suspended in buer for binding assays as described below. 2.4. [3H]MK-801 binding An aliquot (about 0.15 mg protein) of membrane preparations was incubated with 5 nM [3H]MK-801 in 0.5 ml 50 mM Tris-acetate buer (pH 7.4) at 308C for
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
195
Fig. 1. Eects of radical scavengers on inhibition of [3H]MK-801 binding following pretreatment with ferrous chloride. An aliquot of `nonwashed' membranes was incubated with ferrous chloride at three dierent concentrations of 1±100 mM in either the presence or absence of (a) SOD at 200 and 400 units/ml and (b) a-tocopherol at 10 mM, followed by two cycles of washing procedures and subsequent determination of [3H]MK-801 binding under routine conditions (pretreatment). Following two cycles of washing procedures after treatment with ferrous chloride, in addition, membranes were incubated with [3H]MK-801 in either the presence or absence of 10 mM a-tocopherol (addition). Values are from six to nine independent measurements.
30 min unless indicated otherwise (Shuto et al., 1997). Incubation was terminated by the addition of 3 ml icecold buer at 28C and subsequent ®ltration through a Whatman GF/B glass ®ber ®lter under constant vac-
uum of 15 mm Hg. The ®lter was rinsed with 3 ml cold buer at 28C four times within 10 s, and radioactivity retained on the ®lter was measured by a liquid scintillation spectrometer using 5 ml modi®ed Triton-
196
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
Fig. 2. Eects of two dierent phospholipases on inhibition by ferrous chloride of [3H]MK-801 binding. An aliquot of `non-washed' membranes was ®rst treated or not treated with either PLA2 (0.05 units/ml) or PLC (1 unit/ml) in the presence of CaCl2 at 1 mM, followed by two cycles of washing procedures and subsequent second treatment with ferrous chloride at three dierent concentrations from 1 to 100 mM. Following an extra two cycles of washing procedures, [3H]MK-801 binding was determined in either the presence or absence of three distinct agonists including 10 mM Glu, 10 mM Gly and 1 mM SPD under the routine conditions. Values are from four to ®ve separate experiments. *P < 0.05, **P < 0.01, signi®cantly dierent from each control value obtained in membranes treated with vehicle alone. Control binding (fmol/mg protein)ÐNone (no added agonists): none, 156.0217.0; PLA2, 72.329.6; PLC, 241.4221.5. Glu alone: none, 373.9226.1; PLA2, 208.2244.1; PLC, 281.9231.7. Glu/Gly: none, 396.3223.3; PLA2, 231.3252.0; PLC, 215.129.0. Glu/Gly/SPD: none, 738.0242.1; PLA2, 488.62100.1; PLC, 314.5226.8.
toluene scintillant at a counting eciency of 40±42% (Ogita et al., 1986). Nonspeci®c binding of [3H]MK801 was de®ned by the addition of both D-2-amino-5phosphonovaleric and 7-chlorokynurenic acids at 0.1 mM to avoid possible interference with binding by adsorption of the radiolabeled ligand to membrane phospholipids (Suzuki et al., 1993). Protein content was determined by the method of Lowry et al. (1951). In `non-washed' membranes, [3H]MK-801 binding reached equilibrium within 30 min due to the abundance of endogenous agonists such as Glu and Gly. In fact, [3H]MK-801 binding was not signi®cantly potentiated by the addition of both Glu and Gly at maximally eective concentrations in `non-washed' membrane preparations (Enomoto et al., 1992). However, further addition of the polyamine SPD almost doubled binding at equilibrium in `non-washed' membranes. By contrast, [3H]MK-801 binding was low but detectable in membrane preparations extensively
washed by cycles of suspension and subsequent centrifugation as described above. However, extensive washing procedures did not signi®cantly aect pharmacological and biochemical pro®les of [3H]MK-801 binding in synaptic membrane preparations under our experimental conditions as described previously (Enomoto et al., 1992; Ogita et al., 1991). 2.5. Data analysis The concentration of a test compound to inhibit binding by 50% (IC50 value) was calculated according to the Hill plot analysis by the computer program `LOTUS-1-2-3' for Windows `95. Results are all expressed as the mean2SE and the statistical signi®cance was determined by the two-tailed Students' t-test or one way analysis of variance followed by estimation of least signi®cant dierence.
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
197
Table 1 Eects of pretreatment with 2 dierent phospholipases on potencies of ferrous chloride to inhibit [3H]MK-801 binding Addition Pretreatment [None] IC50 (mM) Hill coecient [PLA2] IC50 (mM) Hill coecient [PLC] IC50 (mM) Hill coecient
None
Glu
Glu/Gly
Glu/Gly/SPD
3.520.6 0.8920.07
5.521.2 1.0020.16
10.920.8** 2.0220.25
15.221.5 1.9520.28$
33.328.6#]]] 1.9920.65
55.1215.3#]]] 2.4920.46
54.1214.8#]]] 3.2020.46
63.8212.3#]]] 3.1420.33
35.426.8#]]] 1.7520.52
60.3212.1#]]] 2.2720.43$
74.625.3**#]]] 2.7520.50$
>100.0**#]]] ±
An aliquot of `non-washed' membranes was ®rst treated with either PLA2 (0.05 units/ml) or PLC (1 unit/ml) in the presence of CaCl2 at 1 mM. After two cycles of washing procedures, these memebranes were again treated with ferrous chloride at three dierent concentrations from 1 to 100 mM, followed by same washing procedures and subsequent determination of [3H]MK-801 binding in either the presence or absence of three dierent agonists including 10 mM Glu, 10 mM Gly and 1 mM SPD. Values are from four to ®ve separate experiments. **P < 0.01, signi®cantly dierent from each control value obtained in the absence of added agonists. #]P < 0.05, #]]]P < 0.01, signi®cantly dierent from each control value obtained in membranes treated with vehicle alone. $P < 0.05, signi®cantly dierent from unity.
3. Results 3.1. Irreversible inhibition Pretreatment with ferrous chloride induced more potent inhibition of [3H]MK-801 binding than the addition in a concentration-dependent manner at concentrations of above 1 mM in `non-washed' membranes [IC50 values (mM): addition, 23.421.2; pretreatment, 6.021.6** (P < 0.01)], though binding was markedly decreased by pretreatment and subsequent washing procedures due to removal from membrane preparations of endogenous agonists such as Glu and Gly. Moreover, the addition of iron chelators such as ophenanthroline and deferoxamine, did not signi®cantly aect [3H]MK-801 binding at 100 mM in membranes pretreated with ferrous chloride [IC50 values (mM): none, 2.920.8; o-phenanthroline, 3.321.2; deferoxamine, 5.021.5]. 3.2. Radical scavengers `Non-washed' membranes were incubated with ferrous chloride at dierent concentrations from 1 to 100 mM in either the presence or absence of the superoxide radical scavenger SOD and the peroxyl radical scavenger a-tocopherol, followed by two cycles of washing procedures and subsequent determination of binding. Simultaneous pretreatment with both SOD (200 and 400 units/ml) (Fig. 1a) and 10 mM a-tocopherol (Fig. 1b) did not signi®cantly aect the inhibitory potency of ferrous chloride to inhibit binding. The addition of 10 mM a-tocopherol induced no signi®cant change in [3H]MK-801 binding in membranes which were pretreated with ferrous chloride in the
absence of any added scavengers followed by extensive washing (Fig. 1b). 3.3. Pretreatment with phospholipases `Non-washed' membranes were at ®rst incubated with PLA2 or PLC, followed by two cycles of washing procedures and subsequent treatment with ferrous chloride at dierent concentrations from 1 to 100 mM. Binding of [3H]MK-801 was measured by incubating such pretreated membranes in either the presence or absence of three distinct agonists including Glu, Gly and SPD. The addition of these agonists markedly potentiated binding in control membranes subjected to pretreatment with vehicle and subsequent two cycles of washing [(fmol/mg protein): none, 156.0217.0; Glu alone, 373.9226.1; Glu/Gly, 396.3223.3; Glu/Gly/ SPD, 738.0242.1]. Pretreatment with PLA2 led to a decrease by approximately 50% in binding determined in the absence of any added agonists, while signi®cant potentiation was induced by pretreatment with PLC [(fmol/mg protein): none, 156.0217.0; PLA2, 72.329.6** (P < 0.01); PLC, 241.9221.5** (P < 0.01)]. By contrast, binding was markedly inhibited following pretreatment with both phospholipases when determined in the presence of added agonists. However, the addition of each agonist led to rightward shifts of concentration-response curves for the inhibition by ferrous chloride in membranes pretreated with vehicle alone (Fig. 2). The ®rst pretreatment with PLA2 and PLC signi®cantly prevented the inhibition following the second pretreatment with ferrous chloride in a manner irrespective of the addition of any agonists for determination. Table 1 summarizes these data. In membranes ®rst
198
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
Table 2 Eects of pretreatment with free fatty acids on inhibition by ferrous chloride of [3H]MK-801 binding Free fatty acids [None] [Saturated] Palmitic acid (16:0) Stearic acid (18:0) Arachidic acid (20:0) Behenic acid (22:0) [Unsaturated] Palmitoleic acid (16:1) Oleic acid (18:1) Linoleic acid (18:2) Arachidonic acid (20:4) Docosahexaenoic acid (22:6)
IC50 values (mM)
Hill coecients
5.221.2
0.8020.18
3.721.5 7.522.2 3.721.1 4.921.0
1.3420.54 2.0620.73 1.0820.50 0.8520.33
5.221.9 9.920.5 8.322.1 12.921.0 4.620.6
2.0820.61 0.4820.07$$ 0.5220.17$ 2.0020.50$ 1.1420.35
An aliquot of `non-washed' membranes was ®rst treated or not treated with each fatty acid at 1 mM, followed by two cycles of washing procedures and subsequent treatment with ferrous chloride at three dierent concentrations from 1±100 mM. Following an extra two cycles of washing procedures, [3H]MK-801 binding was determined in the absence of added agonists under the routine conditions. Values are from four to ®ve separate experiments. *P < 0.05, signi®cantly dierent from the control value obtained in membranes not treated with any fatty acids. $P < 0.05, $$P < 0.01, signi®cantly dierent from unity. Control binding (fmol/mg protein): none, 65.2210.6; palmitic acid, 56.924.5; stearic acid, 50.029.6; arachidic acid, 53.229.4; behenic acid, 119.828.7; palmitoleic acid, 47.425.0; oleic acid, 50.424.9; linoleic acid, 51.2211.3; arachidonic acid, 35.725.3; docosahexaenoic acid, 126.428.5.
pretreated with vehicle alone followed by the second treatment with ferrous chloride, the addition of 10 mM Glu alone did not signi®cantly aect the inhibition by ferrous chloride, while the addition of both Glu and Gly at 10 mM signi®cantly attenuated the inhibitory potency of ferrous chloride. Further addition of SPD at 1 mM also diminished the inhibition by ferrous chloride. Moreover, the ®rst pretreatment with PLA2 and PLC induced marked decreases in the potencies of ferrous chloride to inhibit binding determined in the presence of each agonist. Hill coecients were signi®cantly larger than unity in the presence of added agonists in membranes pretreated with PLA2 and PLC in some situations. 3.4. Pretreatment with fatty acids `Non-washed' membranes were ®rst treated with one of saturated and unsaturated free fatty acids at 1 mM followed by 2 cycles of washing procedures, and then treated again with ferrous chloride at dierent concentrations followed by washing. In membranes not treated with ferrous chloride, no signi®cant alterations were observed in binding following the ®rst pretreatment with most saturated fatty acids used, except behenic acid which potentiated binding [(fmol/mg protein): none, 65.2210.6; palmitic acid, 56.924.5; stearic
acid, 50.029.6; arachidic acid, 53.229.4; behenic acid, 119.828.7** (P < 0.01)]. Of unsaturated free fatty acids employed, arachidonic acid signi®cantly inhibited binding in the absence of added agonists, whereas docosahexaenoic acid doubled binding [(fmol/mg protein): none, 65.2210.6; palmitoleic acid, 47.425.0; oleic acid, 50.424.9; linoleic acid, 51.2211.3; arachidonic acid, 35.725.3* (P < 0.05); docosahexaenoic acid, 126.428.5** (P < 0.01)]. All saturated fatty acids failed to aect the inhibition by subsequent pretreatment with ferrous chloride (Table 2). Among unsaturated fatty acids employed, by contrast, both oleic and arachidonic acids were eective in attenuating the inhibition by the second pretreatment with ferrous chloride (Table 2). Hill coecients were signi®cantly smaller than unity for both oleic and linoleic acids but not for other free fatty acids used. 4. Discussion The data cited above raise the possibility that Fe2+ may interfere with opening processes of the native NMDA channel in rat brain. Indeed, elevation of intracellular Ca2+ by NMDA, but not by kainic acid, is suppressed by ferrous sulfate, potassium ferrocyanide and sodium nitroprusside in cultured cerebellar granule cells (Oh and McCaslin, 1995). However, we have previously demonstrated that ferrous chloride inhibits [3H]MK-801 binding to the open NMDA channel in a manner dierent from other Fe2+ -containing compounds such as potassium ferrocyanide and sodium nitroprusside (Shuto et al., 1997). In particular, ferrous chloride rather selectively inhibits [3H]MK-801 binding without markedly aecting binding to the NMDA and Gly recognition domains as well as to ionotropic Glu receptors insensitive to NMDA. By contrast, both potassium ferrocyanide and sodium nitroprusside markedly inhibit binding of [3H]Glu and [3H]DL-(E)-2amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653) to the NMDA recognition domain (Ogita et al., 1998). These data all suggest that ferrous chloride may inhibit [3H]MK-801 binding through mechanisms independent of the recognition processes of the agonists Glu and Gly within the NMDA receptor complex. As membranes were extensively washed following pretreatment with ferrous chloride, the possible direct interaction between iron and [3H]MK-801 is not conceivable. Furthermore, the inhibition by ferrous chloride was not signi®cantly aected by extensive washing in the presence of iron chelators. Therefore, Fe2+ itself could play an additional role critical for mechanisms associated with the in¯ux of Ca2+ across the NMDA channel. Electrophysiological evaluation on Ca2+ currents induced by NMDA of course gives us a key clue for a role of Fe2+ in intracellular signaling cascades
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
triggered by the NMDA receptor complex in membranes. On the other hand, the dependence of lipid peroxidation on iron has been known for many years. Indeed, the ability to inhibit lipid peroxidation is used to identify antioxidants which chelate iron ions or prevent the prooxidant eect by oxidizing Fe2+ to Fe3+ in brain homogenates (Stocks et al., 1974a,b). Further evidence indicates that lipid peroxidation stimulated by ascorbate is dependent on superoxide radicals generated in association with iron in the brain (Andorn et al., 1996). Namely, lipid peroxidation stimulated by ascorbate is not only inhibited by the superoxide radical scavenger SOD in human brain homogenates, but also almost completely abolished by the iron chelator deferoxamine. In addition, a focal injection of ferrous chloride into rat amygdala causes a peroxidative injury of membrane lipids in a manner sensitive to protection by a-tocopherol (Triggs and Willmore, 1994). In homogenates of rat brain, ferrous ammonium sulfate induces lipid peroxidation and protein thiol oxidation which are both inhibited by antioxidants (Linseman et al., 1993). These data all give rise to an idea that Fe2+ induces lipid peroxidation and oxidation of protein thiols in brain membranes in a manner sensitive to protection by scavengers for superoxide and peroxyl radicals. Hence, the Fe2+ -induced inhibition of [3H]MK-801 binding may result from peroxidation of membrane lipids and/or formation of disul®de bonds by oxidation of thiol residues at redox modulatory sites within the NMDA receptor complex (Lei et al., 1992; Lipton et al., 1993; Sullivan et al., 1994). In the present investigation, however, the inhibition by ferrous chloride was not aected by co-application of the potent radical scavengers SOD and a-tocopherol. If lipid peroxidation is really responsible for the inhibition by ferrous ions, either SOD or a-tocopherol should have at least in part modulated the inhibition compared to control in theory. The data thus suggest that the Fe2+ -induced inhibition is not simply mediated by oxidative injuries of membranes as mentioned above. Furthermore, it is also unlikely that ferrous chloride inhibits [3H]MK-801 binding through formation of disul®de bonds by oxidation of thiol residues in the redox modulatory site, because ferrous chloride does not markedly inhibit binding of [3H]Glu and [3H]CGP 39653 to the NMDA recognition domain (Shuto et al., 1997), which is supposed to be induced by oxidation of the redox modulatory site on the NMDA receptor (Ogita et al., 1998). However, the conclusion should await accurate determination of endogenous lipid peroxides and hydroperoxides, but not thiobarbiturate reactive substances, formed in synaptic membranes during incubation with ferrous ions. By contrast, exogenous phospholipases signi®cantly
199
prevented the inhibition of [3H]MK-801 binding by ferrous chloride when treated before the treatment with ferrous chloride. In our hands, both exogenous phospholipases exhibit bell-shaped alterations of [3H]MK-801 binding in the absence of added agonists at a concentration range of 0.001±10 units/ml (Han et al., 1995). One possible interpretation of the prevention is that the Fe2+ -induced inhibition is attenuated following degradation of native phospholipids within brain synaptic membranes. In other words, native phospholipids may be crucial for the inhibition by Fe2+ of binding. Ferrous ions could be released from hemoglobin and transferrin in circulating blood (Yehuda and Youdim, 1988) and thereby act on membrane phospholipids to lead to marked inhibition of the NMDA channel without aecting the non-NMDA channels. Transferrin has ferric ions in its molecule, but is known to release ferrous ions (Yehuda and Youdim, 1988). Another possibility is that the Fe2+ induced inhibition is prevented by certain products derived from membrane phospholipids by the actions of exogenous PLA2 and PLC, including free fatty acids, lisophospholipids and diacylglycerol. In this study, indeed, pretreatment with some unsaturated free fatty acids such as oleic and arachidonic acids, resulted in a slight but statistically signi®cant reduction of the potency of Fe2+ to inhibit [3H]MK-801 binding. The data thus give rise to a proposal that unsaturated free fatty acids may at least in part participate in mechanisms for the prevention by exogenous phospholipases of Fe2+ -induced inhibition of [3H]MK-801 binding following possible liberation from native phospholipids in synaptic membranes. The possibility that other products from membrane phospholipids may mediate the inhibition by Fe2+ in an unidenti®ed manner, however, is not ruled out so far. Indeed, both exogenous phospholipases were much more potent than each unsaturated free fatty acid in preventing the inhibition by Fe2+ . Finally, Fe2+ seems to inhibit [3H]MK-801 binding to the open NMDA channel under regulations by membrane phospholipids within synaptic membranes. An alternative explanation is alterations by ferrous ions as well as phospholipases in the composition of membrane phospholipids. In fact, ¯uorescence polarization analysis reveals developmental alterations of membrane order (Rauch and Hitzemann, 1986) as well as membrane ¯uidity (Hitzemann and Harris, 1984) due to changes in lipid composition in rat brain. These changes in lipid composition could be responsible for prevention by phospholipases against the inhibition by Fe2+ of [3H]MK-801 binding in membrane preparations as seen in this study. Membrane ¯uidity could be also modulated by unsaturated free fatty acids and lisophospholipids either of which is liberated from membrane phospholipids by the actions of PLA2 and
200
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
PLC. The fact that both oleic and arachidonic acids attenuated the inhibition is not unfavorable to an idea that membrane ¯uidity may be one important determinant of modulation by ferrous chloride of the opening of the NMDA channel. However, the data from experiments using other saturated and unsaturated fatty acids do not always give support to the idea. Because of a direct interaction of SPD with particular phospholipids such as phosphatidylserine (Suzuki et al., 1993; Yoneda et al., 1991), in addition, SPD may in¯uence the Fe2+ -induced inhibition through membrane phospholipids. Hill coecients signi®cantly larger and smaller than unity suggest positive and negative cooperativity between ferrous ions and [3H]MK-801 binding under the conditions employed, respectively. As mentioned above, Fe2+ has the ability to inhibit 3 [ H]MK-801 binding in rat brain synaptic membranes under control by membrane phospholipids, in a manner that seems independent of both lipid peroxidation and oxidation of the redox modulatory site within the NMDA receptor. However, it still remains unclear where Fe2+ directly attacks in synaptic membranes which for instance contain so many candidates including phospholipids, subunit proteins composing the diverse NMDA channels (Hollmann and Heinemann, 1994; Nakanishi and Masu, 1994; Monyer et al., 1994) and modulatory proteins for opening processes of the NMDA channels. The irreversible inhibition would be accounted for by taking into consideration the modulation of some unidenti®ed membrane constituents in response to Fe2+ . The irreversibility could also argue in favor of the proposal that the inhibition by Fe2+ may be operative under particular pathological conditions, such as cerebral and subarachnoid hemorrhage, which lead to a drastic increase in the extracellular Fe2+ concentration in the vicinity of heterogeneous NMDA channels. The NMDA receptor could be under control by delicate balancing between the agonist Glu and the modulator Fe2+ which are both released during those neurodegenerative disorders. It thus appears that Fe2+ plays a critical role in mechanisms associated with opening processes of the NMDA channel through irreversible alteration of unidenti®ed membrane constituents in rat brain. The search for target components is undoubtedly of great bene®t for the therapy and treatment of a variety of neurological disorders associated with malfunction of the NMDA receptor in human brain. Acknowledgements This work was supported in part by Grants-in-Aid for Scienti®c Research to Y.Y. from the Ministry of Education, Science, Sports and Culture, Japan.
References Agostinho, P., Duarte, C.B., Oliveira, C.R., 1996. Activity of ionotropic glutamate receptors in retinal cells: eect of ascorbate/ Fe2+ -induced oxidative stress. J. Neurochem. 67, 1153±1163. Andorn, A.C., Britton, R.S., Bacon, B.R., 1996. Ascorbate-stimulated lipid peroxidation in human brain is dependent on iron but not on hydroxyl radical. J. Neurochem. 67, 717±722. Benkovic, S.A., Connor, J.R., 1993. Ferritin, transferrin, and iron in selected regions of the adult and aged rat brain. J. Comp. Neurol. 45, 97±113. Bradbury, M.W.B., 1997. Transport of iron in the blood-brain-cerebrospinal ¯uid system. J. Neurochem. 69, 443±454. Choi, D.W., 1988. Glutamate neurotoxicity and disease of the nervous system. Neuron 1, 623±634. Collingridge, G.L., Singer, W., 1990. Excitatory amino acid receptors and synaptic plasticity. Trends Pharmacol. Sci. 11, 290±296. Cotman, C.W., Monaghan, D.T., Ganong, A.H., 1988. Excitatory amino acid transmission: NMDA receptors and Hebb-type synaptic plasticity. Ann. Rev. Neurosci. 11, 61±80. Ehlers, M.D., Zhang, S., Bernhardt, J.P., Huganir, R.L., 1996. Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84, 745±755. Enomoto, R., Ogita, K., Han, D., Yoneda, Y., 1992. Dierential modulation by divalent cations of [3H]MK-801 binding in brain synaptic membranes. J. Neurochem. 59, 473±481. Floyd, R.A., Carney, J.M., 1991. Age in¯uence on oxidative events during brain ischemia/reperfusion. Arch. Gerontol. Geriatr. 12, 155±177. Gerlach, M., Ben-Shachar, D., Riederer, P., Youdim, M.B.H., 1994. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J. Neurochem. 63, 793±807. Hall, E.D., Braughler, J.M., 1993. Free radical in CNS injuries. In: Waxman, S.G. (Ed.), Molecular and Cellular Approaches to the Treatment of Neurological Disease. Raven Press, New York, pp. 81±105. Halliwell, B., 1992. Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609±1623. Han, D., Ogita, K., Yoneda, Y., 1995. Potentiation by calcium ions of [3H]MK-801 binding to an ion channel associated with the Nmethyl-D-aspartate receptor complex in rat brain. Neurochem. Int. 26, 59±68. Hill, J.M., Switzer, R.C., 1984. The regional distribution and cellular localisation of iron in the rat brain. Neuroscience 11, 595±603. Hitzemann, R.J., Harris, R.A., 1984. Developmental changes in synaptic membrane ¯uidity: a comparison of 1,6-diphenyl-1,3,5hexatriene (DPH) and 1-[4-(trimethylamino)-phenyl]-6-phenyl1,3,5-hexatriene (TMA-DPH). Brain Res. 316, 113±120. Hollmann, M., Heinemann, S., 1994. Cloned glutamate receptors. Ann. Rev. Neurosci. 23, 31±108. Lei, S.Z., Pan, Z-H., Aggarwal, S.K., Cen, H-S.V., Hartman, J., Sucher, N.J., Lipton, S.A., 1992. Eect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 8, 1087±1099. Linseman, K.L., Larson, P., Braughler, J.M., McCall, J.M., 1993. Iron-initiated tissue oxidation: lipid peroxidation, vitamin E destruction and protein thiol oxidation. Inhibit by a novel antioxidant, U-78517F. Biochem. Pharmacol. 45, 1477±1482. Lipton, S.A., Rosenberg, P.A., 1994. Mechanisms of disease: excitatory amino acids as a ®nal common pathway for neurologic disorders. New Engl. J. Med. 330, 613±622. Lipton, S.A., Choi, Y-B., Pan, Z-H., Lei, S.Z., Stamler, J.S., 1993. A redox-based mechanism for the neuroprotective and neurodestructive eects of nitric oxide and related nitroso-compounds. Nature 364, 626±632. Lodge, D., Collingridge, G., 1991. The pharmacology of excitatory
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201 amino acids. A TiPS special report. Trends Pharmacol. Sci. special issue, 1±89. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265±275. Monyer, H., Burnashev, N., Laurie, D.J., Sakmann, B., Seeburg, P., 1994. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529± 540. Nakanishi, S., Masu, M., 1994. Molecular diversity and functions of glutamate receptors. Ann. Rev. Biophys. Biomol. Struct. 23, 319± 348. Ogita, K., Shuto, M., Yoneda, Y., 1998. Nitric oxide-independent inhibition by sodium nitroprusside of the native N-methyl-D-aspartate recognition domain in a manner dierent from that by potassium ferrocyanide. Neurochem. Int. 33, 1±9. Ogita, K., Suzuki, T., Pingping, Z., Yoneda, Y., 1991. Inhibition by calmodulin antagonists of [3H]MK-801 binding in brain synaptic membranes. J. Neurochem. 59, 1008±1016. Ogita, K., Kitago, T., Nakamuta, H., Fukuda, Y., Koida, M., Ogawa, Y., Yoneda, Y., 1986. Glutathione-induced inhibition of Na+-independent and -dependent bindings of L-[3H]glutamate in rat brain. Life Sci. 39, 2411±2418. Oh, S., McCaslin, P.P., 1995. The iron component of sodium nitroprusside blocks NMDA-induced glutamate accumulation and intracellular Ca2+ elevation. Neurochem. Res. 20, 779±784. Rauch, S.L., Hitzemann, R.J., 1986. Developmental changes in synaptic membrane order: a comparison of regions in the rat brain. Brain Res. 391, 221±227. Riederer, P., So®c, E., Rausch, W-D., Schmidt, B., Reynolds, G.P., Jellinger, K., Youdim, M.B.H., 1989. Transition metals, ferritin, glutathione, and ascorbic acid in Parkinsonian brains. J. Neurochem. 52, 515±520. Shuto, M., Ogita, K., Minami, T., Maeda, H., Yoneda, Y., 1997. Inhibition of [3H]MK-801 binding by ferrous (II) but not ferric (III) ions in a manner dierent from that by sodium nitroprusside (II) in rat brain synaptic membranes. J. Neurochem. 69, 744±752.
201
Stocks, J., Gutteridge, J.M.C., Sharp, R.J., Dormandy, T.L., 1974a. Assay using brain homogenate for measuring the antioxidant activity of biological ¯uids. Clin. Sci. Mol. Med. 47, 215±222. Stocks, J., Gutteridge, J.M.C., Sharp, R.J., Dormandy, T.L., 1974b. The inhibition of lipid autooxidation by human serum and its relationships to serum proteins and alpha-tocopherol. Clin. Sci. Mol. Med. 47, 223±233. Sullivan, J.M., Traynelis, S.F., Chen, H-S.V., Escobar, W., Heinemann, S.F., Lipton, S.A., 1994. Identi®cation of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor. Neuron 13, 929±936. Suzuki, T., Ogita, K., Yoneda, Y., 1993. Potentiation by polyamine of an interaction of noncompetitive antagonists at the N-methylD-aspartate receptor ionophore complex with phosphatidylserine. Neurochem. Int. 23, 427±440. Triggs, W.J., Willmore, L.J., 1994. Eect of [dl]-alpha-tocopherol on FeCl2-induced lipid peroxidation in rat amygdala. Neurosci. Lett. 180, 33±36. Yehuda, S., Youdim, M.B.H., 1988. Brain iron de®ciency. Biochemistry and behavior. In: Youdim, M.B.H. (Ed.), Brain Iron, Neurochemical and Behavioral Aspects. Taylor & Francis, London, pp. 89±114. Yoneda, Y., Ogita, K., 1989. Microbial methodological artifacts in [3H]glutamate receptor binding assays. Anal. Biochem. 177, 250± 255. Yoneda, Y., Ogita, K., Enomoto, R., Suzuki, T., Kito, S., 1991. Identi®cation and characterization of speci®c binding sites of [3H]spermidine in synaptic membranes of rat brain. Brain Res. 563, 17±27. Youdim, M.B.H., Ben-Shachar, D., Riederer, P., 1993. The possible role of iron in the etiopathology of Parkinson's disease. Mov. Disord. 8, 1±12. Zhang, J-R., Scherch, H.M., Hall, E.D., 1996. Direct measurement of lipid hydroperoxides in iron-dependent spinal neuronal injury. J. Neurochem. 66, 355±361.
Preventive eects of exogenous phospholipases on inhibition by ferrous ions of [3H]MK-801 binding in rat brain synaptic membranes Kiyokazu Ogita, Makoto Shuto, Takayuki Manabe, Nobuyuki Kuramoto, Yukio Yoneda* Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-0101, Japan
Abstract Prior treatment with ferrous chloride led to marked inhibition of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 mM in rat brain synaptic membranes. Both phospholipases A2 and C signi®cantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor a-tocopherol aected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated free fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were ®rst treated with fatty acids, followed by the second treatment with ferrous chloride. These results suggest that membrane phospholipids may be at least in part responsible for interference by ferrous ions with opening processes of the native NMDA channel through molecular mechanisms associated with the liberation of unsaturated free fatty acids in rat brain. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Excitatory amino acid receptors are thought to play a key role in mechanisms underlying important physiological functions of the brain such as learning and memory (Collingridge and Singer, 1990; Cotman et al., 1988), in addition to crisis of a variety of acute neurological and chronic neurodegenerative disorders. The latter includes hypoxic-ishemic brain injury, trauma, epilepsy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and AIDS dementia (Choi, 1988; Lipton and Rosenberg, 1994). Early pharmacological studies have revealed that an ionotropic subclass sensitive to N-methyl-D-aspartic acid (NMDA) functions as a receptor ionophore complex consisting * Corresponding author. Tel.: +81-720-66-3109; fax: +81-720-663109. E-mail address: [email protected] (Y. Yoneda)
of at least four distinct domains: (a) an ion channel domain highly permeable to Ca2+ , (b) an NMDA recognition domain with high anity for the endogenous agonist L-glutamic acid (Glu), (c) a glycine (Gly) recognition domain with anity for co-agonists including Gly and D-serine and (d) a polyamine recognition domain with several inde®nite properties. Moreover, opening of the NMDA channel is interfered by endogenous cations including Mg2+ , Zn2+ and H+, in voltage-dependent and independent manners. The NMDA channel is radiolabeled by [3H](+)-5-methyl10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) which only gains access to the binding site located within the channel when it is opened (Lodge and Collingridge, 1991). Moreover, increased intracellular Ca2+ is supposed to be responsible for feedback inhibition of opening processes of the NMDA channel through calmodulin linked to an intracellular C-terminal region within the principal subunit
0197-0186/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 8 ) 0 0 0 8 7 - 4
194
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
NMDAR1 (Ehlers et al., 1996). By contrast, dierent calmodulin antagonists all inhibit (Ogita et al., 1991), but Ca2+ ions stimulate (Enomoto et al., 1992; Han et al., 1995) binding of [3H]MK-801 to the open NMDA channel in rat brain synaptic membranes. In addition to regulation by these endogenous cations, the in vitro addition of Fe2+ but not Fe3+ inhibits [3H]MK-801 binding through mechanisms entirely dierent from those underlying the inhibition by other Fe2+ -containing compounds including sodium nitroprusside and potassium ferrocyanide (Shuto et al., 1997). Iron is an important constituent in the brain at a substantially high concentration as compared with any other metals (Yehuda and Youdim, 1988). Among dierent brain regions, for instance, iron is enriched at a relatively high concentration in the basal ganglia, red nucleus and dentate nucleus (Bradbury, 1997; Hill and Switzer, 1984; Riederer et al., 1989). Iron is shown to play a critical role in the pathogenesis of particular neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, in association with oxidative stress through the generation of oxygen free radicals (Gerlach et al., 1994), which are supposed to be responsible for mediating neuronal injuries associated with iron by way of lipid peroxidation of cell membranes (Floyd and Carney, 1991; Hall and Braughler, 1993; Halliwell, 1992). Indeed, Fe2+ markedly facilitates formation of lipid hydroperoxides with a concomitant loss of cellular viability in cultured spinal neurons (Zhang et al., 1996). Therefore, lipid peroxidation by iron could ignite cascade processes for crisis of a variety of neurological disorders associated with aging (Youdim et al., 1993) which is shown to induce increased accumulation of iron in particular regions of rat brain (Benkovic and Connor, 1993). However, an opposite proposal that ascorbate/Fe2+ -induced oxidative stress leads to increased activities of the NMDA receptor, such as [3H]g-aminobutyric acid release, Na+ in¯ux and [3H]MK-801 binding in cultured retinal cells is available in the literature (Agostinho et al., 1996). Accordingly, the present study was undertaken to investigate possible mechanisms for the inhibition by ferrous chloride of [3H]MK-801 binding to a native ion channel associated with the NMDA receptor in rat brain synaptic membranes. 2. Materials and methods 2.1. Materials [3H]MK-801 ([3-3H]MK-801, 740 GBq/mmol) was purchased from NEN/DuPont (Boston, MA, USA). Arachidonic, behenic, docosahexaenoic, linoleic, oleic and palmitoeic acids, phospholipase A2 (PLA2), phospholipase C (PLC), spermidine (SPD), superoxide dis-
mutase (SOD) and a-tocopherol were all provided by Sigma Chemical (St. Louis, MA, USA). Stearic acid was purchased from Doosan Serdary Research Laboratories (NJ, USA). Arachidic acid, ferrous chloride and palmitic acid were supplied by Wako Chemical (Osaka, Japan). Other chemicals used were all of the highest purity commercially available. All solutions were freshly prepared immediately before each use. 2.2. Membrane preparation Crude synaptic membrane fractions obtained from whole brains (including cerebellum) of adult male Wistar rats weighing 200±250 g were washed once by suspension in 40 volumes of 50 mM Tris-acetate buer (pH 7.4) using a Physcotron homogenizer at a setting no. 6 for 1 min at 48C, followed by centrifugation at 50,000 g for 30 min, as described previously (Enomoto et al., 1992). Resultant pellets were suspended in 8 volumes of the same buer and stored at ÿ808C until use. On the day of the experiment, these frozen suspensions were thawed at room temperature and directly used for binding assays described below as `non-washed' membrane preparations. Buers and any other solutions used in the present study were all ®ltered each time before use through a nitrocellulose membrane ®lter with a pore size of 450 nm to avoid possible microbial contamination (Yoneda and Ogita, 1989). 2.3. Pretreatment `Non-washed' membranes were incubated with either phospholipases or free fatty acids at 308C for 30 min in 50 mM Tris-acetate buer (pH 7.4) at an approximate protein concentration of 0.2 mg protein/ml, followed by centrifugation at 50,000 g for 30 min. The resultant pellets were again suspended in the same volume of buer followed by centrifugation. Pellets thus obtained were suspended in the same volume of buer, and the suspensions were again incubated with ferrous chloride at dierent concentrations for 30 min at 308C, followed by two cycles of washing procedures as described above. In addition, `non-washed' membranes were pretreated with ferrous chloride at dierent concentrations in either the presence or absence of 10 mM a-tocopherol and SOD at 200 and 400 units/ml, followed by two cycles of washing procedures, as needed. The ®nal pellets thus obtained were suspended in buer for binding assays as described below. 2.4. [3H]MK-801 binding An aliquot (about 0.15 mg protein) of membrane preparations was incubated with 5 nM [3H]MK-801 in 0.5 ml 50 mM Tris-acetate buer (pH 7.4) at 308C for
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
195
Fig. 1. Eects of radical scavengers on inhibition of [3H]MK-801 binding following pretreatment with ferrous chloride. An aliquot of `nonwashed' membranes was incubated with ferrous chloride at three dierent concentrations of 1±100 mM in either the presence or absence of (a) SOD at 200 and 400 units/ml and (b) a-tocopherol at 10 mM, followed by two cycles of washing procedures and subsequent determination of [3H]MK-801 binding under routine conditions (pretreatment). Following two cycles of washing procedures after treatment with ferrous chloride, in addition, membranes were incubated with [3H]MK-801 in either the presence or absence of 10 mM a-tocopherol (addition). Values are from six to nine independent measurements.
30 min unless indicated otherwise (Shuto et al., 1997). Incubation was terminated by the addition of 3 ml icecold buer at 28C and subsequent ®ltration through a Whatman GF/B glass ®ber ®lter under constant vac-
uum of 15 mm Hg. The ®lter was rinsed with 3 ml cold buer at 28C four times within 10 s, and radioactivity retained on the ®lter was measured by a liquid scintillation spectrometer using 5 ml modi®ed Triton-
196
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
Fig. 2. Eects of two dierent phospholipases on inhibition by ferrous chloride of [3H]MK-801 binding. An aliquot of `non-washed' membranes was ®rst treated or not treated with either PLA2 (0.05 units/ml) or PLC (1 unit/ml) in the presence of CaCl2 at 1 mM, followed by two cycles of washing procedures and subsequent second treatment with ferrous chloride at three dierent concentrations from 1 to 100 mM. Following an extra two cycles of washing procedures, [3H]MK-801 binding was determined in either the presence or absence of three distinct agonists including 10 mM Glu, 10 mM Gly and 1 mM SPD under the routine conditions. Values are from four to ®ve separate experiments. *P < 0.05, **P < 0.01, signi®cantly dierent from each control value obtained in membranes treated with vehicle alone. Control binding (fmol/mg protein)ÐNone (no added agonists): none, 156.0217.0; PLA2, 72.329.6; PLC, 241.4221.5. Glu alone: none, 373.9226.1; PLA2, 208.2244.1; PLC, 281.9231.7. Glu/Gly: none, 396.3223.3; PLA2, 231.3252.0; PLC, 215.129.0. Glu/Gly/SPD: none, 738.0242.1; PLA2, 488.62100.1; PLC, 314.5226.8.
toluene scintillant at a counting eciency of 40±42% (Ogita et al., 1986). Nonspeci®c binding of [3H]MK801 was de®ned by the addition of both D-2-amino-5phosphonovaleric and 7-chlorokynurenic acids at 0.1 mM to avoid possible interference with binding by adsorption of the radiolabeled ligand to membrane phospholipids (Suzuki et al., 1993). Protein content was determined by the method of Lowry et al. (1951). In `non-washed' membranes, [3H]MK-801 binding reached equilibrium within 30 min due to the abundance of endogenous agonists such as Glu and Gly. In fact, [3H]MK-801 binding was not signi®cantly potentiated by the addition of both Glu and Gly at maximally eective concentrations in `non-washed' membrane preparations (Enomoto et al., 1992). However, further addition of the polyamine SPD almost doubled binding at equilibrium in `non-washed' membranes. By contrast, [3H]MK-801 binding was low but detectable in membrane preparations extensively
washed by cycles of suspension and subsequent centrifugation as described above. However, extensive washing procedures did not signi®cantly aect pharmacological and biochemical pro®les of [3H]MK-801 binding in synaptic membrane preparations under our experimental conditions as described previously (Enomoto et al., 1992; Ogita et al., 1991). 2.5. Data analysis The concentration of a test compound to inhibit binding by 50% (IC50 value) was calculated according to the Hill plot analysis by the computer program `LOTUS-1-2-3' for Windows `95. Results are all expressed as the mean2SE and the statistical signi®cance was determined by the two-tailed Students' t-test or one way analysis of variance followed by estimation of least signi®cant dierence.
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
197
Table 1 Eects of pretreatment with 2 dierent phospholipases on potencies of ferrous chloride to inhibit [3H]MK-801 binding Addition Pretreatment [None] IC50 (mM) Hill coecient [PLA2] IC50 (mM) Hill coecient [PLC] IC50 (mM) Hill coecient
None
Glu
Glu/Gly
Glu/Gly/SPD
3.520.6 0.8920.07
5.521.2 1.0020.16
10.920.8** 2.0220.25
15.221.5 1.9520.28$
33.328.6#]]] 1.9920.65
55.1215.3#]]] 2.4920.46
54.1214.8#]]] 3.2020.46
63.8212.3#]]] 3.1420.33
35.426.8#]]] 1.7520.52
60.3212.1#]]] 2.2720.43$
74.625.3**#]]] 2.7520.50$
>100.0**#]]] ±
An aliquot of `non-washed' membranes was ®rst treated with either PLA2 (0.05 units/ml) or PLC (1 unit/ml) in the presence of CaCl2 at 1 mM. After two cycles of washing procedures, these memebranes were again treated with ferrous chloride at three dierent concentrations from 1 to 100 mM, followed by same washing procedures and subsequent determination of [3H]MK-801 binding in either the presence or absence of three dierent agonists including 10 mM Glu, 10 mM Gly and 1 mM SPD. Values are from four to ®ve separate experiments. **P < 0.01, signi®cantly dierent from each control value obtained in the absence of added agonists. #]P < 0.05, #]]]P < 0.01, signi®cantly dierent from each control value obtained in membranes treated with vehicle alone. $P < 0.05, signi®cantly dierent from unity.
3. Results 3.1. Irreversible inhibition Pretreatment with ferrous chloride induced more potent inhibition of [3H]MK-801 binding than the addition in a concentration-dependent manner at concentrations of above 1 mM in `non-washed' membranes [IC50 values (mM): addition, 23.421.2; pretreatment, 6.021.6** (P < 0.01)], though binding was markedly decreased by pretreatment and subsequent washing procedures due to removal from membrane preparations of endogenous agonists such as Glu and Gly. Moreover, the addition of iron chelators such as ophenanthroline and deferoxamine, did not signi®cantly aect [3H]MK-801 binding at 100 mM in membranes pretreated with ferrous chloride [IC50 values (mM): none, 2.920.8; o-phenanthroline, 3.321.2; deferoxamine, 5.021.5]. 3.2. Radical scavengers `Non-washed' membranes were incubated with ferrous chloride at dierent concentrations from 1 to 100 mM in either the presence or absence of the superoxide radical scavenger SOD and the peroxyl radical scavenger a-tocopherol, followed by two cycles of washing procedures and subsequent determination of binding. Simultaneous pretreatment with both SOD (200 and 400 units/ml) (Fig. 1a) and 10 mM a-tocopherol (Fig. 1b) did not signi®cantly aect the inhibitory potency of ferrous chloride to inhibit binding. The addition of 10 mM a-tocopherol induced no signi®cant change in [3H]MK-801 binding in membranes which were pretreated with ferrous chloride in the
absence of any added scavengers followed by extensive washing (Fig. 1b). 3.3. Pretreatment with phospholipases `Non-washed' membranes were at ®rst incubated with PLA2 or PLC, followed by two cycles of washing procedures and subsequent treatment with ferrous chloride at dierent concentrations from 1 to 100 mM. Binding of [3H]MK-801 was measured by incubating such pretreated membranes in either the presence or absence of three distinct agonists including Glu, Gly and SPD. The addition of these agonists markedly potentiated binding in control membranes subjected to pretreatment with vehicle and subsequent two cycles of washing [(fmol/mg protein): none, 156.0217.0; Glu alone, 373.9226.1; Glu/Gly, 396.3223.3; Glu/Gly/ SPD, 738.0242.1]. Pretreatment with PLA2 led to a decrease by approximately 50% in binding determined in the absence of any added agonists, while signi®cant potentiation was induced by pretreatment with PLC [(fmol/mg protein): none, 156.0217.0; PLA2, 72.329.6** (P < 0.01); PLC, 241.9221.5** (P < 0.01)]. By contrast, binding was markedly inhibited following pretreatment with both phospholipases when determined in the presence of added agonists. However, the addition of each agonist led to rightward shifts of concentration-response curves for the inhibition by ferrous chloride in membranes pretreated with vehicle alone (Fig. 2). The ®rst pretreatment with PLA2 and PLC signi®cantly prevented the inhibition following the second pretreatment with ferrous chloride in a manner irrespective of the addition of any agonists for determination. Table 1 summarizes these data. In membranes ®rst
198
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
Table 2 Eects of pretreatment with free fatty acids on inhibition by ferrous chloride of [3H]MK-801 binding Free fatty acids [None] [Saturated] Palmitic acid (16:0) Stearic acid (18:0) Arachidic acid (20:0) Behenic acid (22:0) [Unsaturated] Palmitoleic acid (16:1) Oleic acid (18:1) Linoleic acid (18:2) Arachidonic acid (20:4) Docosahexaenoic acid (22:6)
IC50 values (mM)
Hill coecients
5.221.2
0.8020.18
3.721.5 7.522.2 3.721.1 4.921.0
1.3420.54 2.0620.73 1.0820.50 0.8520.33
5.221.9 9.920.5 8.322.1 12.921.0 4.620.6
2.0820.61 0.4820.07$$ 0.5220.17$ 2.0020.50$ 1.1420.35
An aliquot of `non-washed' membranes was ®rst treated or not treated with each fatty acid at 1 mM, followed by two cycles of washing procedures and subsequent treatment with ferrous chloride at three dierent concentrations from 1±100 mM. Following an extra two cycles of washing procedures, [3H]MK-801 binding was determined in the absence of added agonists under the routine conditions. Values are from four to ®ve separate experiments. *P < 0.05, signi®cantly dierent from the control value obtained in membranes not treated with any fatty acids. $P < 0.05, $$P < 0.01, signi®cantly dierent from unity. Control binding (fmol/mg protein): none, 65.2210.6; palmitic acid, 56.924.5; stearic acid, 50.029.6; arachidic acid, 53.229.4; behenic acid, 119.828.7; palmitoleic acid, 47.425.0; oleic acid, 50.424.9; linoleic acid, 51.2211.3; arachidonic acid, 35.725.3; docosahexaenoic acid, 126.428.5.
pretreated with vehicle alone followed by the second treatment with ferrous chloride, the addition of 10 mM Glu alone did not signi®cantly aect the inhibition by ferrous chloride, while the addition of both Glu and Gly at 10 mM signi®cantly attenuated the inhibitory potency of ferrous chloride. Further addition of SPD at 1 mM also diminished the inhibition by ferrous chloride. Moreover, the ®rst pretreatment with PLA2 and PLC induced marked decreases in the potencies of ferrous chloride to inhibit binding determined in the presence of each agonist. Hill coecients were signi®cantly larger than unity in the presence of added agonists in membranes pretreated with PLA2 and PLC in some situations. 3.4. Pretreatment with fatty acids `Non-washed' membranes were ®rst treated with one of saturated and unsaturated free fatty acids at 1 mM followed by 2 cycles of washing procedures, and then treated again with ferrous chloride at dierent concentrations followed by washing. In membranes not treated with ferrous chloride, no signi®cant alterations were observed in binding following the ®rst pretreatment with most saturated fatty acids used, except behenic acid which potentiated binding [(fmol/mg protein): none, 65.2210.6; palmitic acid, 56.924.5; stearic
acid, 50.029.6; arachidic acid, 53.229.4; behenic acid, 119.828.7** (P < 0.01)]. Of unsaturated free fatty acids employed, arachidonic acid signi®cantly inhibited binding in the absence of added agonists, whereas docosahexaenoic acid doubled binding [(fmol/mg protein): none, 65.2210.6; palmitoleic acid, 47.425.0; oleic acid, 50.424.9; linoleic acid, 51.2211.3; arachidonic acid, 35.725.3* (P < 0.05); docosahexaenoic acid, 126.428.5** (P < 0.01)]. All saturated fatty acids failed to aect the inhibition by subsequent pretreatment with ferrous chloride (Table 2). Among unsaturated fatty acids employed, by contrast, both oleic and arachidonic acids were eective in attenuating the inhibition by the second pretreatment with ferrous chloride (Table 2). Hill coecients were signi®cantly smaller than unity for both oleic and linoleic acids but not for other free fatty acids used. 4. Discussion The data cited above raise the possibility that Fe2+ may interfere with opening processes of the native NMDA channel in rat brain. Indeed, elevation of intracellular Ca2+ by NMDA, but not by kainic acid, is suppressed by ferrous sulfate, potassium ferrocyanide and sodium nitroprusside in cultured cerebellar granule cells (Oh and McCaslin, 1995). However, we have previously demonstrated that ferrous chloride inhibits [3H]MK-801 binding to the open NMDA channel in a manner dierent from other Fe2+ -containing compounds such as potassium ferrocyanide and sodium nitroprusside (Shuto et al., 1997). In particular, ferrous chloride rather selectively inhibits [3H]MK-801 binding without markedly aecting binding to the NMDA and Gly recognition domains as well as to ionotropic Glu receptors insensitive to NMDA. By contrast, both potassium ferrocyanide and sodium nitroprusside markedly inhibit binding of [3H]Glu and [3H]DL-(E)-2amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653) to the NMDA recognition domain (Ogita et al., 1998). These data all suggest that ferrous chloride may inhibit [3H]MK-801 binding through mechanisms independent of the recognition processes of the agonists Glu and Gly within the NMDA receptor complex. As membranes were extensively washed following pretreatment with ferrous chloride, the possible direct interaction between iron and [3H]MK-801 is not conceivable. Furthermore, the inhibition by ferrous chloride was not signi®cantly aected by extensive washing in the presence of iron chelators. Therefore, Fe2+ itself could play an additional role critical for mechanisms associated with the in¯ux of Ca2+ across the NMDA channel. Electrophysiological evaluation on Ca2+ currents induced by NMDA of course gives us a key clue for a role of Fe2+ in intracellular signaling cascades
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
triggered by the NMDA receptor complex in membranes. On the other hand, the dependence of lipid peroxidation on iron has been known for many years. Indeed, the ability to inhibit lipid peroxidation is used to identify antioxidants which chelate iron ions or prevent the prooxidant eect by oxidizing Fe2+ to Fe3+ in brain homogenates (Stocks et al., 1974a,b). Further evidence indicates that lipid peroxidation stimulated by ascorbate is dependent on superoxide radicals generated in association with iron in the brain (Andorn et al., 1996). Namely, lipid peroxidation stimulated by ascorbate is not only inhibited by the superoxide radical scavenger SOD in human brain homogenates, but also almost completely abolished by the iron chelator deferoxamine. In addition, a focal injection of ferrous chloride into rat amygdala causes a peroxidative injury of membrane lipids in a manner sensitive to protection by a-tocopherol (Triggs and Willmore, 1994). In homogenates of rat brain, ferrous ammonium sulfate induces lipid peroxidation and protein thiol oxidation which are both inhibited by antioxidants (Linseman et al., 1993). These data all give rise to an idea that Fe2+ induces lipid peroxidation and oxidation of protein thiols in brain membranes in a manner sensitive to protection by scavengers for superoxide and peroxyl radicals. Hence, the Fe2+ -induced inhibition of [3H]MK-801 binding may result from peroxidation of membrane lipids and/or formation of disul®de bonds by oxidation of thiol residues at redox modulatory sites within the NMDA receptor complex (Lei et al., 1992; Lipton et al., 1993; Sullivan et al., 1994). In the present investigation, however, the inhibition by ferrous chloride was not aected by co-application of the potent radical scavengers SOD and a-tocopherol. If lipid peroxidation is really responsible for the inhibition by ferrous ions, either SOD or a-tocopherol should have at least in part modulated the inhibition compared to control in theory. The data thus suggest that the Fe2+ -induced inhibition is not simply mediated by oxidative injuries of membranes as mentioned above. Furthermore, it is also unlikely that ferrous chloride inhibits [3H]MK-801 binding through formation of disul®de bonds by oxidation of thiol residues in the redox modulatory site, because ferrous chloride does not markedly inhibit binding of [3H]Glu and [3H]CGP 39653 to the NMDA recognition domain (Shuto et al., 1997), which is supposed to be induced by oxidation of the redox modulatory site on the NMDA receptor (Ogita et al., 1998). However, the conclusion should await accurate determination of endogenous lipid peroxides and hydroperoxides, but not thiobarbiturate reactive substances, formed in synaptic membranes during incubation with ferrous ions. By contrast, exogenous phospholipases signi®cantly
199
prevented the inhibition of [3H]MK-801 binding by ferrous chloride when treated before the treatment with ferrous chloride. In our hands, both exogenous phospholipases exhibit bell-shaped alterations of [3H]MK-801 binding in the absence of added agonists at a concentration range of 0.001±10 units/ml (Han et al., 1995). One possible interpretation of the prevention is that the Fe2+ -induced inhibition is attenuated following degradation of native phospholipids within brain synaptic membranes. In other words, native phospholipids may be crucial for the inhibition by Fe2+ of binding. Ferrous ions could be released from hemoglobin and transferrin in circulating blood (Yehuda and Youdim, 1988) and thereby act on membrane phospholipids to lead to marked inhibition of the NMDA channel without aecting the non-NMDA channels. Transferrin has ferric ions in its molecule, but is known to release ferrous ions (Yehuda and Youdim, 1988). Another possibility is that the Fe2+ induced inhibition is prevented by certain products derived from membrane phospholipids by the actions of exogenous PLA2 and PLC, including free fatty acids, lisophospholipids and diacylglycerol. In this study, indeed, pretreatment with some unsaturated free fatty acids such as oleic and arachidonic acids, resulted in a slight but statistically signi®cant reduction of the potency of Fe2+ to inhibit [3H]MK-801 binding. The data thus give rise to a proposal that unsaturated free fatty acids may at least in part participate in mechanisms for the prevention by exogenous phospholipases of Fe2+ -induced inhibition of [3H]MK-801 binding following possible liberation from native phospholipids in synaptic membranes. The possibility that other products from membrane phospholipids may mediate the inhibition by Fe2+ in an unidenti®ed manner, however, is not ruled out so far. Indeed, both exogenous phospholipases were much more potent than each unsaturated free fatty acid in preventing the inhibition by Fe2+ . Finally, Fe2+ seems to inhibit [3H]MK-801 binding to the open NMDA channel under regulations by membrane phospholipids within synaptic membranes. An alternative explanation is alterations by ferrous ions as well as phospholipases in the composition of membrane phospholipids. In fact, ¯uorescence polarization analysis reveals developmental alterations of membrane order (Rauch and Hitzemann, 1986) as well as membrane ¯uidity (Hitzemann and Harris, 1984) due to changes in lipid composition in rat brain. These changes in lipid composition could be responsible for prevention by phospholipases against the inhibition by Fe2+ of [3H]MK-801 binding in membrane preparations as seen in this study. Membrane ¯uidity could be also modulated by unsaturated free fatty acids and lisophospholipids either of which is liberated from membrane phospholipids by the actions of PLA2 and
200
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201
PLC. The fact that both oleic and arachidonic acids attenuated the inhibition is not unfavorable to an idea that membrane ¯uidity may be one important determinant of modulation by ferrous chloride of the opening of the NMDA channel. However, the data from experiments using other saturated and unsaturated fatty acids do not always give support to the idea. Because of a direct interaction of SPD with particular phospholipids such as phosphatidylserine (Suzuki et al., 1993; Yoneda et al., 1991), in addition, SPD may in¯uence the Fe2+ -induced inhibition through membrane phospholipids. Hill coecients signi®cantly larger and smaller than unity suggest positive and negative cooperativity between ferrous ions and [3H]MK-801 binding under the conditions employed, respectively. As mentioned above, Fe2+ has the ability to inhibit 3 [ H]MK-801 binding in rat brain synaptic membranes under control by membrane phospholipids, in a manner that seems independent of both lipid peroxidation and oxidation of the redox modulatory site within the NMDA receptor. However, it still remains unclear where Fe2+ directly attacks in synaptic membranes which for instance contain so many candidates including phospholipids, subunit proteins composing the diverse NMDA channels (Hollmann and Heinemann, 1994; Nakanishi and Masu, 1994; Monyer et al., 1994) and modulatory proteins for opening processes of the NMDA channels. The irreversible inhibition would be accounted for by taking into consideration the modulation of some unidenti®ed membrane constituents in response to Fe2+ . The irreversibility could also argue in favor of the proposal that the inhibition by Fe2+ may be operative under particular pathological conditions, such as cerebral and subarachnoid hemorrhage, which lead to a drastic increase in the extracellular Fe2+ concentration in the vicinity of heterogeneous NMDA channels. The NMDA receptor could be under control by delicate balancing between the agonist Glu and the modulator Fe2+ which are both released during those neurodegenerative disorders. It thus appears that Fe2+ plays a critical role in mechanisms associated with opening processes of the NMDA channel through irreversible alteration of unidenti®ed membrane constituents in rat brain. The search for target components is undoubtedly of great bene®t for the therapy and treatment of a variety of neurological disorders associated with malfunction of the NMDA receptor in human brain. Acknowledgements This work was supported in part by Grants-in-Aid for Scienti®c Research to Y.Y. from the Ministry of Education, Science, Sports and Culture, Japan.
References Agostinho, P., Duarte, C.B., Oliveira, C.R., 1996. Activity of ionotropic glutamate receptors in retinal cells: eect of ascorbate/ Fe2+ -induced oxidative stress. J. Neurochem. 67, 1153±1163. Andorn, A.C., Britton, R.S., Bacon, B.R., 1996. Ascorbate-stimulated lipid peroxidation in human brain is dependent on iron but not on hydroxyl radical. J. Neurochem. 67, 717±722. Benkovic, S.A., Connor, J.R., 1993. Ferritin, transferrin, and iron in selected regions of the adult and aged rat brain. J. Comp. Neurol. 45, 97±113. Bradbury, M.W.B., 1997. Transport of iron in the blood-brain-cerebrospinal ¯uid system. J. Neurochem. 69, 443±454. Choi, D.W., 1988. Glutamate neurotoxicity and disease of the nervous system. Neuron 1, 623±634. Collingridge, G.L., Singer, W., 1990. Excitatory amino acid receptors and synaptic plasticity. Trends Pharmacol. Sci. 11, 290±296. Cotman, C.W., Monaghan, D.T., Ganong, A.H., 1988. Excitatory amino acid transmission: NMDA receptors and Hebb-type synaptic plasticity. Ann. Rev. Neurosci. 11, 61±80. Ehlers, M.D., Zhang, S., Bernhardt, J.P., Huganir, R.L., 1996. Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84, 745±755. Enomoto, R., Ogita, K., Han, D., Yoneda, Y., 1992. Dierential modulation by divalent cations of [3H]MK-801 binding in brain synaptic membranes. J. Neurochem. 59, 473±481. Floyd, R.A., Carney, J.M., 1991. Age in¯uence on oxidative events during brain ischemia/reperfusion. Arch. Gerontol. Geriatr. 12, 155±177. Gerlach, M., Ben-Shachar, D., Riederer, P., Youdim, M.B.H., 1994. Altered brain metabolism of iron as a cause of neurodegenerative diseases? J. Neurochem. 63, 793±807. Hall, E.D., Braughler, J.M., 1993. Free radical in CNS injuries. In: Waxman, S.G. (Ed.), Molecular and Cellular Approaches to the Treatment of Neurological Disease. Raven Press, New York, pp. 81±105. Halliwell, B., 1992. Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609±1623. Han, D., Ogita, K., Yoneda, Y., 1995. Potentiation by calcium ions of [3H]MK-801 binding to an ion channel associated with the Nmethyl-D-aspartate receptor complex in rat brain. Neurochem. Int. 26, 59±68. Hill, J.M., Switzer, R.C., 1984. The regional distribution and cellular localisation of iron in the rat brain. Neuroscience 11, 595±603. Hitzemann, R.J., Harris, R.A., 1984. Developmental changes in synaptic membrane ¯uidity: a comparison of 1,6-diphenyl-1,3,5hexatriene (DPH) and 1-[4-(trimethylamino)-phenyl]-6-phenyl1,3,5-hexatriene (TMA-DPH). Brain Res. 316, 113±120. Hollmann, M., Heinemann, S., 1994. Cloned glutamate receptors. Ann. Rev. Neurosci. 23, 31±108. Lei, S.Z., Pan, Z-H., Aggarwal, S.K., Cen, H-S.V., Hartman, J., Sucher, N.J., Lipton, S.A., 1992. Eect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 8, 1087±1099. Linseman, K.L., Larson, P., Braughler, J.M., McCall, J.M., 1993. Iron-initiated tissue oxidation: lipid peroxidation, vitamin E destruction and protein thiol oxidation. Inhibit by a novel antioxidant, U-78517F. Biochem. Pharmacol. 45, 1477±1482. Lipton, S.A., Rosenberg, P.A., 1994. Mechanisms of disease: excitatory amino acids as a ®nal common pathway for neurologic disorders. New Engl. J. Med. 330, 613±622. Lipton, S.A., Choi, Y-B., Pan, Z-H., Lei, S.Z., Stamler, J.S., 1993. A redox-based mechanism for the neuroprotective and neurodestructive eects of nitric oxide and related nitroso-compounds. Nature 364, 626±632. Lodge, D., Collingridge, G., 1991. The pharmacology of excitatory
K. Ogita et al. / Neurochemistry International 34 (1999) 193±201 amino acids. A TiPS special report. Trends Pharmacol. Sci. special issue, 1±89. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265±275. Monyer, H., Burnashev, N., Laurie, D.J., Sakmann, B., Seeburg, P., 1994. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529± 540. Nakanishi, S., Masu, M., 1994. Molecular diversity and functions of glutamate receptors. Ann. Rev. Biophys. Biomol. Struct. 23, 319± 348. Ogita, K., Shuto, M., Yoneda, Y., 1998. Nitric oxide-independent inhibition by sodium nitroprusside of the native N-methyl-D-aspartate recognition domain in a manner dierent from that by potassium ferrocyanide. Neurochem. Int. 33, 1±9. Ogita, K., Suzuki, T., Pingping, Z., Yoneda, Y., 1991. Inhibition by calmodulin antagonists of [3H]MK-801 binding in brain synaptic membranes. J. Neurochem. 59, 1008±1016. Ogita, K., Kitago, T., Nakamuta, H., Fukuda, Y., Koida, M., Ogawa, Y., Yoneda, Y., 1986. Glutathione-induced inhibition of Na+-independent and -dependent bindings of L-[3H]glutamate in rat brain. Life Sci. 39, 2411±2418. Oh, S., McCaslin, P.P., 1995. The iron component of sodium nitroprusside blocks NMDA-induced glutamate accumulation and intracellular Ca2+ elevation. Neurochem. Res. 20, 779±784. Rauch, S.L., Hitzemann, R.J., 1986. Developmental changes in synaptic membrane order: a comparison of regions in the rat brain. Brain Res. 391, 221±227. Riederer, P., So®c, E., Rausch, W-D., Schmidt, B., Reynolds, G.P., Jellinger, K., Youdim, M.B.H., 1989. Transition metals, ferritin, glutathione, and ascorbic acid in Parkinsonian brains. J. Neurochem. 52, 515±520. Shuto, M., Ogita, K., Minami, T., Maeda, H., Yoneda, Y., 1997. Inhibition of [3H]MK-801 binding by ferrous (II) but not ferric (III) ions in a manner dierent from that by sodium nitroprusside (II) in rat brain synaptic membranes. J. Neurochem. 69, 744±752.
201
Stocks, J., Gutteridge, J.M.C., Sharp, R.J., Dormandy, T.L., 1974a. Assay using brain homogenate for measuring the antioxidant activity of biological ¯uids. Clin. Sci. Mol. Med. 47, 215±222. Stocks, J., Gutteridge, J.M.C., Sharp, R.J., Dormandy, T.L., 1974b. The inhibition of lipid autooxidation by human serum and its relationships to serum proteins and alpha-tocopherol. Clin. Sci. Mol. Med. 47, 223±233. Sullivan, J.M., Traynelis, S.F., Chen, H-S.V., Escobar, W., Heinemann, S.F., Lipton, S.A., 1994. Identi®cation of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor. Neuron 13, 929±936. Suzuki, T., Ogita, K., Yoneda, Y., 1993. Potentiation by polyamine of an interaction of noncompetitive antagonists at the N-methylD-aspartate receptor ionophore complex with phosphatidylserine. Neurochem. Int. 23, 427±440. Triggs, W.J., Willmore, L.J., 1994. Eect of [dl]-alpha-tocopherol on FeCl2-induced lipid peroxidation in rat amygdala. Neurosci. Lett. 180, 33±36. Yehuda, S., Youdim, M.B.H., 1988. Brain iron de®ciency. Biochemistry and behavior. In: Youdim, M.B.H. (Ed.), Brain Iron, Neurochemical and Behavioral Aspects. Taylor & Francis, London, pp. 89±114. Yoneda, Y., Ogita, K., 1989. Microbial methodological artifacts in [3H]glutamate receptor binding assays. Anal. Biochem. 177, 250± 255. Yoneda, Y., Ogita, K., Enomoto, R., Suzuki, T., Kito, S., 1991. Identi®cation and characterization of speci®c binding sites of [3H]spermidine in synaptic membranes of rat brain. Brain Res. 563, 17±27. Youdim, M.B.H., Ben-Shachar, D., Riederer, P., 1993. The possible role of iron in the etiopathology of Parkinson's disease. Mov. Disord. 8, 1±12. Zhang, J-R., Scherch, H.M., Hall, E.D., 1996. Direct measurement of lipid hydroperoxides in iron-dependent spinal neuronal injury. J. Neurochem. 66, 355±361.